One of the major challenges of proteomics is the sheer complexity of biomolecule samples, such as blood serum or cell extract. Typical blood samples could contain more than 10,000 different protein species, with concentrations varying over 9 orders of magnitude. Such diversity of proteins, as well as their huge concentration ranges, poses a formidable challenge for sample preparation in proteomics.
Conventional protein analysis techniques, based on multidimensional separation steps and mass spectrometry (MS), fall short because of the limited separation peak capacity (up to ˜3000) and dynamic range of detection (˜104). Microfluidic biomolecule analysis systems (so-called μTAS) hold promise for automated biomolecule processing. Various biomolecule separation and purification steps, as well as chemical reaction and amplification have been miniaturized on a microchip, demonstrating orders of magnitude faster sample separation and processing. In addition, microfluidic integration of two different separation steps into a multidimensional separation device has been demonstrated. However, most microfluidic separation and sample processing devices suffers from the critical issue of sample volume mismatch. Microfluidic devices are very efficient in handling and processing 1 pL˜1 nL of sample fluids, but most biomolecule samples are available or handled in a liquid volume larger than 1 μL. Therefore, microchip-based separation techniques often analyze only a small fraction of available samples, which significantly limits the overall detection sensitivity. In proteomics, this problem is exacerbated by the fact that information-rich signaling molecules (cytokines and biomarkers, e.g.) are present only in trace concentrations (nM˜pM range), and there is no signal amplification technique such as polymerase chain reaction (PCR) for proteins and peptides.
What is needed is an efficient sample concentrator, which can take typical sample volume of microliters or more and concentrate molecules into a smaller volume so that it can be separated and detected much more sensitively. Several strategies are currently available to provide sample preconcentration in liquid, including field-amplified sample stacking (FAS), isotachophoresis (ITP), electrokinetic trapping, micellar electrokinetic sweeping, chromatographic preconcentration, and membrane preconcentration. Many of these techniques are originally developed for capillary electrophoresis, and require special buffer arrangements and/or reagents. Efficiency of chromatographic and filtration-based preconcentration techniques depends on the hydrophobicity and the size of the target molecules.
Electrokinetic trapping is another means for such charged biomolecule concentration. When applying an electric field across an ion-selective membrane, a charge-depletion region is developed, which in combination with tangential flow (either pressure-driven or electroosmosis-driven), can concentrate the charged analytes inside a channel. Currently, however, the fabrication of such devices is cumbersome and complex, since the integration of sufficiently thin (˜5 um) ion-selective membranes into the device has been challenging. Thin Nafion membranes are easily breakable and handling requires extreme care since the membrane can be easily wrapped around itself, confounding planar device fabrication methods.
Another attempt at planar devices sandwiched a thin ion-selective membrane between two planar microchips, each chip containing a microchannel, however this led to imperfect sealing of the device, resulting in gap formation around the membrane and thereby current leakage.